No. 3] Proc. Jpn. Acad., Ser. B 95 (2019) 111

Review Molecular mechanisms of coupling to voltage sensors in voltage-evoked cellular signals

† By Yasushi OKAMURA*1,*2, and Yoshifumi OKOCHI*1

(Communicated by Masanori OTSUKA, M.J.A.)

Abstract: The voltage sensor domain (VSD) has long been studied as a unique domain intrinsic to voltage-gated ion channels (VGICs). Within VGICs, the VSD is tightly coupled to the pore-gate domain (PGD) in diverse ways suitable for its specific function in each physiological context, including generation, muscle contraction and relaxation, hormone and neurotransmitter secretion, and cardiac pacemaking. However, some VSD-containing lack a PGD. Voltage-sensing phosphatase contains a cytoplasmic phosphoinositide phosphatase with similarity to phosphatase and tensin homolog (PTEN). Hv1, a voltage-gated proton channel, also lacks a PGD. Within Hv1, the VSD operates as a voltage sensor, gate, and pore for both proton sensing and permeation. Hv1 has a C-terminal coiled coil that mediates dimerization for cooperative gating. Recent progress in the structural biology of VGICs and VSD proteins provides insights into the principles of VSD coupling conserved among these proteins as well as the hierarchy of organization for voltage-evoked cell signaling.

Keywords: voltage sensor, membrane potential, phosphoinositide, proton, gating

Membrane excitation plays fundamental roles in these ion channels were identified through studies the functions of neurons, muscle, endocrine cells, and involving biochemistry, molecular genetics,4) and D D 5),6) electric organs. Na and K conductances through molecular cloning. Proteins comprising Nav,Kv, D the membrane, mediated by voltage-gated Na and and Cav channels share a common architecture that D K ion (Nav and Kv) channels, respectively, were includes a voltage sensor domain (VSD) consisting of the first elements shown to underlie the membrane four transmembrane helices and a pore-gate domain excitability of nerves.1) Later, voltage-gated Ca2D (PGD) consisting of two transmembrane helices with (Cav) channels were identified in muscle, neurons, an intervening turret region (Fig. 1). These voltage- and endocrine cells.2),3) The molecular correlates of gated ion channels (VGICs), together with related

1 D D * Department of Physiology, Graduate School of Medicine, Kcv: virus-derived K channel; Kir: inward rectifier K ;Kv: Osaka University, Suita, Japan. voltage-gated KD; MPO: myeloperoxidase; NADPH: nicotinamide 2 D * Graduate School of Frontier Bioscience, Osaka adenine dinucleotide phosphate; Nav: voltage-gated Na ; NHE: University, Suita, Japan. sodium proton exchanger; PBM: phosphoinositide-binding † Correspondence should be addressed: Y. Okamura, motif; PD: phosphatase domain; PGD: pore-gate domain; Department of Physiology, Graduate School of Medicine and PHD: pleckstrin homology domain; PI: phosphoinositide; PS: Graduate School of Frontier Bioscience, Osaka University, Suita, pregnenolone sulfate; PtdIns(3,4)P2: phosphatidylinositol-3,4- Osaka 565-0871, Japan (e-mail: [email protected]. bisphosphate; PtdIns(3,4,5)P3: phosphatidylinositol-3,4,5-tri- ac.jp). sphosphate; PtdIns(3,5)P2: phosphatidylinositol-3,5-bisphosphate; Abbreviations: Anap: 3-(6-acetylnaphthalen-2-ylamino)- PtdIns(4,5)P2: phosphatidylinositol-4,5-bisphosphate; PtdIns4P: 2-aminopropanoic acid; CatSper: cation channel of sperm; Cav: phosphatidylinositol-4-monophosphate; PTEN: phosphatase and voltage-gated Ca2D; CNG: cyclic nucleotide-gated; cryo-EM: tensin homolog (deleted on ten); ROS: reactive cryoelectron microscopy; EPR: electron paramagnetic resonance; oxygen species; RyR: ; SNARE complex: soluble FRET: fluorescence resonance energy transfer; GEVI: - NSF attachment protein receptor complex; TAPP1: tandem PH encoded voltage indicator; GFP: green fluorescent protein; H2O2: domain containing protein 1; TPC: two-pore channel; TRIC: hydrogen peroxide; HCN channel: hyperpolarization-activated, trimeric intracellular channel; TRP: transient receptor potential; cyclic nucleotide-gated channel; HOCl: hypochlorous acid; VCF: voltage clamp fluorometry; VGIC: voltage-gated ion HVCN1: hydrogen voltage-gated channel 1 (gene name for Hv1, channel; VSD: voltage sensor domain; VSLD: voltage-sensor like voltage-gated proton channel); HVRP1: Hv1-related protein 1; domain; VSP: voltage-sensing phosphatase. doi: 10.2183/pjab.95.010 ©2019 The Japan Academy 112 Y. OKAMURA and Y. OKOCHI [Vol. 95,

a Voltage-sensor domain Pore-gate domain x 4 : tetramer Voltage-gated or 2 or 4 homologous repeats in single subunit Phosphatase domain C2 domain VSP x 1: monomer

Coiled coil

Hv1/VSOP x 2: dimer

Pore-gate Voltage-sensor b domain domain S6 S4 S5 + + + + S1 + S2 S3

Fig. 1. Scheme of the membrane topologies of VGIC and two VSD-containing proteins. a. VSP and Hv1. VGIC contains the voltage- sensor domain (VSD) and the pore-gate domain (PGD). Most of VGICs contain N-terminal and C-terminal cytoplasmic domains which are important for subunit assembly and gating (not specifically illustrated here). VSP has the transmembrane VSD and the cytoplasmic enzyme region which consists of the phosphatase domain and C2 domain. There is a short linker between the VSD and PD (called VSD-PD linker) (not illustrated here). Transmembrane region of Hv1, the voltage-gated proton channel, corresponds to the VSD of VGIC and VSP. Hv1 contains the cytoplasmic coiled coil region which is critical for dimer formation and gating. b. Illustration of one unit of VSD (consisting of four transmembrane helices) and PGD of domain-swapped type VGIC. Helical linker connects the two domains. See Fig. 8 for domain-nonswapped type VGIC. proteins, such as hyperpolarization-activated, cyclic more recently, with single particle cryo-electron nucleotide-gated channels (HCN channels) and two- microscopy (cryo-EM) (Fig. 2).8) One remarkable pore channels (TPCs), are all classified as members finding from recent studies of the structures of VGICs 7) of the VGIC superfamily. In Kv and HCN channels, is that the coupling of the VSD to the PGD is diverse one subunit contains a single VSD and PGD, and and optimized for the physiological functions of four subunits assemble into one channel. In Nav and each individual class of VGIC. Moreover, in several Cav, one channel is formed by a long polypeptide atypical VSD-containing proteins, the coupling of consisting of four homologous repeats, each of which the VSD is not for driving a PGD. In voltage-sensing contains a single VSD and PGD. In TPCs, one phosphatases, for example, the VSD is coupled to channel is formed by two homologous subunits, each the enzyme and regulates phosphatase activity. In containing two repeats. Transient receptor potential the voltage-gated proton channel, Hv1, which lacks (TRP) channels are ion channels transducing multi- a PGD, the VSD operates as an ion permeation modal chemical and physical stimuli, such as stretch, pathway as well as a voltage sensor, and two VSDs temperature change, and oxygen concentration, into interact with each other for cooperative gating. How intracellular signals. The transmembrane regions of protons selectively permeate through the VSD in Hv1 TRP channels have a molecular architecture that remains a mystery. In addition, cyclic nucleotide- resembles that of VGICs, consisting of a voltage- gated (CNG) channels and many TRP channels have sensor like domain (VSLD) and a PGD. immobile VSDs. The protein structures of most VGIC species In this article, we first summarize the structure– have been solved using X-ray crystallography and, function relationship and molecular mechanisms of No. 3] Principles and diversity of voltage sensor domain proteins 113

Kv1.2-Kv2.1 chimera

NavPaS TPC1 Cav1.1

HCN1 Slo1

Ci-VSP mHv1cc

Fig. 2. Gallery of protein structures of VGICs and VSP (taken from ref. 157). Kv1.2–Kv2.1 chimera: human voltage-gated (PDB ID: 2R9R), TPC1: plant two-pore cation channel (PDB ID: 5E1J), Cav1.1: human skeletal muscle-type voltage-gated (PDB ID: 5GJV), NavPaS: insect voltage-gated (PDB ID: 5X0M), HCN1: human hyperpolarization- activated cyclic nucleotide-gated channel (PDB ID: 5U6O), Slo1: large conductance calcium-activated voltage-gated potassium channel from Aplysia (PDB ID: 5TJ6), Ci-VSP: sea squirt voltage-sensing phosphatase (full length model based on coordinates from PDB ID: 3AWF and PDB ID: 4G7V), mHv1cc: mouse voltage-gated proton channel in a chimeric form containing part of S2–S3 from Ci-VSP and the coiled coil structure of GCN4 (dimer model based on coordinates from PDB ID: 3WKV). NavPaS, Kv1.2–Kv2.1 chimera and Slo1 have four VSDs within a tetramer. TPC1 has four VSDs within a dimer. Ci-VSP has a single VSD. mHv1cc has two VSDs within a dimer. coupling between the VSD and PGD in VGICs. sensing membrane voltage and is highly conserved Then, we describe VSD-effector coupling and VSD- among VGICs. It consists of four transmembrane VSD coupling in two VSD-containing proteins, helices, S1–S4, with S4 showing a signature pattern voltage-sensing phosphatase (VSP) and Hv1, respec- of amino acid alignment: positively charged residues tively, both of which do not contain a canonical aligned with two intervening hydrophobic residues PGD. We also state more emerging functions of the (Fig. 3a).9) The positive charges in S4 are counter- VSD-like region in other proteins and the hierarchy balanced by multiple negative charges on acidic of voltage signals. Finally, future directions of residues in the other three helices (S1–S3) to form research are discussed. salt bridges that are periodically aligned, like a ladder (Fig. 3b). This ladder-like alignment of salt bridges 1. The VSD in VGICs spans a constricted highly hydrophobic region. A The VSD is a protein structure critical for hydrophobic residue, phenylalanine, in S2 is highly 114 Y. OKAMURA and Y. OKOCHI [Vol. 95,

a Kv1.2/2.1 VVQIFRIMRILRIFKLSRH306 Ci-VSP LVVLARLLRVVRLARIFYSH 239 mHv1 LLRLWRVARIINGIIISVKT 218

b

Fig. 3. Amino acid sequence of S4 (a) and the X-ray crystal structure of the VSD of Ci-VSP with its membrane topology (b: taken from ref. 157).

conserved among VGICs, except in some rare The crystal structure shows the S3–S4 region cases.10) This residue functions to separate two protruding to the external side of the protein in the aqueous crevices, thereby providing a charge transfer solvent environment. This structure led to the center for the positive charges on S4 to translocate. hypothesis that parts of S3 and S4 and the Sensing an electric field across the membrane,11),12) intervening extracellular loop (S3–S4 loop) forms a S4 undergoes translation or helical motion against paddle-like structure and undergoes substantial the other three helices. The charge displacement movement from the hydrophobic to the aqueous achieved through this voltage-induced conforma- environment (“paddle” model).17) Later, however, the tional change in the VSD can be measured with an solved structure of the human Kv channel showed the electrophysiological method as a macroscopic tran- position of S4 within the VSD and suggested that S4 13),14) sient current (gating currents). in the crystal structure of KvAP was dislocated to the The PGD consists of two transmembrane helices outside as a result of the crystal packing. Never- (S5 and S6) and an intervening loop and contains two theless, the paddle model stimulated several bio- major structures that regulate ion permeation. The physical studies of S4 motion.18),19) Various models loop between S5 and S6 forms a selectivity filter near of S4 motion, including the transporter model, in the extracellular exit of the pore. This determines which there is only minor helical motion of S4, and which ions can permeate through the pore. Bundled simple translation of S4 through other parts of the crossing of four sets of S5 and S6 near the intra- VSD, were proposed.20) These models differ with cellular exit of the pore forms the gate structure. A respect to the distance and direction of S4 motion. part of S6 on the side of the membrane closer to the Later, the solved atomic structures of VGICs and cytoplasm contains a kinked region, which is critical biophysical measurements led to the consensus idea for regulating gating.15) that the movement of S4 is characterized by a How the VSD senses changes in transmembrane mixture of translational and helical screw motions.20) potential and what motion is induced have long been A study of long time scale (9 ms), all-atom molecular hot issues. The first X-ray crystal structure of a dynamics simulations of the Kv channel upon a VGIC was that of the Kv channel from the change in membrane potential also support such 16) 21) archeabacterium Aeropyrum pernix, called KvAP. motion of S4. No. 3] Principles and diversity of voltage sensor domain proteins 115

Domain-non-swapped type Domain-swapped type

Kv10 Kv1.2 Kv11 Kv7.1 HCN Nav Slo1 Cav Slo2 TPC1

Kv10.1 (EMD-8215) Kv1.2-1.2 chimera (2R9R)

Fig. 4. Two categories of VGICs dependent on VSD-PGD organization: domain-swapped vs. domain-nonswapped. A figure from ref. 37 is used.

mitted to the gate through the linker and C-terminal – 2. Domain domain interaction in VGICs part of S6. It should be noted that in these types As is evident from their names, VGICs are of VGICs the cytoplasmic region is not domain- classified based on their biophysical nature, includ- swapped. For example, the T1 domain of Kv1, ing ion selectivity, which is rigidly dictated by the which is known to be involved in tetrameric structure of the PGD and the direction of the assembly but not in regulating channel gating, is membrane potential that activates channel opening. not domain-swapped. A subunit-assembly domain Most VGICs are activated by depolarization, containing a coiled-coil motif in the C-terminal although HCN channels are activated by hyper- cytoplasmic region of Kv7 is also not domain- 25) polarization. In many types of Kv,Nav, and Cav swapped. channels,22)–24) as well as TPCs, which are endo- The critical functions of the S4–S5 linker and somal/lysosomal voltage-gated cation channels, the C-terminal part of S6 in VSD-PGD coupling were VSD is “domain-swapped” with the PGD. In other established in a number of studies, two of which are words, the VSD is linked to the PGD within the seminal. In one of these studies, voltage dependence same subunit (in Kv) or repeat (in Nav and Cav), but was conferred to the pH-gated potassium channel is located next to the PGD in a neighboring subunit from the soil bacteria Streptomyces lividans, which is (or repeat) (Fig. 4, right panel). The linker between called the KcsA channel (a channel that lacks a VSD) the VSD and PGD (called the S4–S5 linker) of one by constructing a chimeric protein with Kv1 (which subunit runs beneath the pore-forming helix of contains a VSD). In that case, the transfer of not only another subunit before reaching the PGD of the the VSD but also the S4–S5 linker and C-terminal same subunit. The S4–S5 linker is a helix forming a part of S6 helix from Kv1 was necessary to confer cuff around the S4 helix that also makes contact voltage sensitivity on the KcsA channel.26) The with the C-terminal part of S6. This category of second seminal study was an examination of ther- channels with a VSD characterized as being domain- mo-energetics with mutant cycle analysis in the fruit 27) swapped includes most classically studied VGICs, flyKv1 channel ( channel). Three critical including Nav,Kv,andCav channels, which are amino acids were identified for coupling: two charged primarily activated by membrane depolarization. residues in the S4–S5 linker (Arg-394, Glu-395: Conformational changes in the VSD can be trans- numbers correspond to the Shaker sequence) and 116 Y. OKAMURA and Y. OKOCHI [Vol. 95, tryptophan (Tyr-485) in S6. Modeling based on the aromatic residues.35) In addition, a noncanonical crystal structure of Kv1.2/2.1 predicts these residues mode of electromechanical coupling involving inter- form a complex and suggests the upward motion of faces between two transmembrane helices, S4 and S5, S4 causes motion in the complex involving both the was recently reported for the Shaker channel.36) Such S4–S5 linker and the C-terminal end of S6 helix that helix–helix interactions may underlie cooperativity induces pore opening in the same subunit (Fig. 7a). among subunits during gating. 37) An all-atom molecular dynamics simulation of the Kv In some VGIC channels, including Kv10, 38) 39) 10) 40) channel response to a membrane potential change Kv11, HCN, Slo1, and Slo2, S4 is packed also supported this motion of the S4–S5 linker.21) with S5 in the same subunit (i.e., the domain is not Moreover, recent cryo-EM studies of eukaryotic Nav swapped), and the S4–S5 linker is much shorter than channels enabled comparison of the structures of in authentic VGICs (Fig. 4, left panel). Channels the S4–S5 linker in different channel states, which belonging to the domain-nonswapped group have supports the idea of lever lifting motion of the linker been solved using either X-ray crystallography or 28),29) upon the motion of S4. cryo-EM. In Kv10.1, coupling between the VSD and Of note, in some VGICs the coupling between PGD does not require physical linkage between the the VSD and PGD is also chemically regulated. In two domains, unlike in domain-swapped VGICs the KCNQ1 (Kv7.1)/KCNE1 channel complex, (Fig. 4, Fig. 8). This is illustrated by the fact that which underlies slow outward currents in cardiac upon cutting the channel into two polypeptides at muscle, it is known that phosphoinositide (PI) the linker between the VSD and PGD, the channel regulates channel activity. Cui’s group showed that reassembles to function normally as a Kv chan- 41),42) PtdIns(4,5)P2 binds to the S4–S5 linker of KCNQ1 nel. Splitting the linker does not dramatically to ensure coupling between the VSD and PGD perturb the channel structure, as abnormal gating is (Fig. 8b),30),31) and a recent cryo-EM structure of reversed by replacement of a single amino acid near KCNQ1 is consistent with that model.32) The TPC1 the split site. It has been suggested that slight motion channel is a multimodal sodium channel activated by in S4 can compress the S5 helix, triggering the motion both membrane depolarization and binding of in S6, the pore-forming helix. 2D PtdIns(3,5)P2, which is most abundant in endo- In Slo1, the large-conductance, Ca - and somes/lysosomes, where TPC1 is selectively ex- voltage-activated KD channel, S6 is positioned close pressed. An atomic structure of the mammalian to a cytoplasmic ring-like structure, which is con- TPC1 channel in complex with PtdIns(3,5)P2 nected to the sensing domains for divalent cations 2D showed that PtdIns(3,5)P2 docks near the S4–S5 (Ca bowl and RCK domain for binding divalent linker, facing part of S6 close to the cytoplasm and cations). In the HCN channel, S6 is positioned close the N-terminus of S3.33) to the cyclic nucleotide binding domain, which is In Nav,Kv,andCav channels, the VSD of one required for binding cyclic nucleotides. In these subunit is spatially closer to the PGD of a neighboring domain-nonswapped VGICs, the cytoplasmic region subunit than the PGD in the same subunit. This is domain-swapped, in contrast to domain-swapped raises the natural question, does the VSD of one VGICs. Notably, many domain-nonswapped VGICs subunit interact with the PGD of a neighboring are activated not only by a membrane potential subunit? In Kv1.2/2.1, the external end of S1 is change but also by intracellular small molecules, situated next to the external side of S5. This Ca2D ions (Slo1), NaD ions (in Slo2.2), cAMP (in 2D interaction probably ensures stabilization of the HCN), or Ca -calmodulin complex (in Kv10). This entire channel structure during dynamic motion of raises the possibility that the domain-swapped the mobile part (S3–S4) mediating voltage-dependent architecture in the cytoplasmic region together with gating.34) Potential intersubunit interaction between the nondomain swapped architecture of the VSD the VSD and PGD has also been observed within the and PGD may be favorable for dual regulation of S6 KCNQ1/KCNE1 complex, where a phenylalanine in motion by both the VSD and the cytoplasmic sensor S4 interacts with a phenylalanine in S5 in the presence structure that binds small molecules. However, this of KCNE1. This slows depolarization-activated pore idea may be premature, because the TPC channel, opening without significantly affecting the motion which is a domain-swapped VGIC, is regulated by 2D D of S4. Amino acid replacement experiments support intracellular Ca , and the Erg K (Kv11) channel speculation that such interaction depends on steric is regulated mainly by membrane potential, not hindrance between the two bulky side chains of the through binding small molecules. No. 3] Principles and diversity of voltage sensor domain proteins 117

To date, the significance of swapping and residues (Fig. 3). A study of the crystal structures of nonswapping domain interactions between VSD the VSD from Ci-VSP suggests that upon a change in and PGD in the gating mechanisms of VGICs membrane potential, there is a simple upward helical remains unclear. motion of S4 without significant motion change in the other helices.46) Single molecule imaging suggests 3. VSD regulates intrinsic enzyme of a VSP VSP is a monomer. However, a recent study by the The VSD was long thought to be unique to same group showed that VSP proteins can assemble VGICs. However, in 2005, we serendipitously dis- into dimers, depending on their density on the cell covered an exceptional case in a bioinformatics surface,47),48) which is consistent with the results of analysis of the genome of a sea squirt, Ciona an earlier fluorescence resonance energy transfer intestinalis. We found a VSP has a VSD that is (FRET) study of fluorescent proteins.49) connected not to a PGD but to a cytoplasmic Membrane depolarization activates the enzyme structure with homology to tumor suppressor phos- activity of PI phosphatases.50) Unlike with VGICs, phatase (phosphatase and tensin homolog quantitative measurement of the readout of VSD [PTEN]).43) Using a similar bioinformatics approach, action is not straightforward in VSP, where the we also identified Hv1, the voltage-gated proton outcome is enzymatic activity not ion conduc- channel, as another example of a VSD lacking a tion. VSP dephosphorylates PtdIns(3,4,5)P3, 51)–53) PGD. The VSD of Hv1 functions as both a voltage PtdIns(3,4)P2, and PtdIns(4,5)P2. The robust sensing apparatus and a proton-permeable pore. 5-phosphate phosphatase activity of VSP toward 3-1. A single VSD couples with the enzyme PtdIns(4,5)P2 is in contrast to PTEN, which is to change the PI profile in voltage-sensing highly selective for the 3-phosphate position of 54) phosphatase. In all VGICs, multiple VSDs work PtdIns(3,4,5)P3 and PtdIns(3,4)P2. The enzymatic together to regulate a pore in the center of activity of VSP toward PtdIns(4,5)P2 can be homologous units (subunits or repeats). In a survey monitored through electrophysiological measurement of ion channel-related from the whole genome of the activity of coexpressed ion channels that of Ciona intestinalis, several genes were not catego- depend on the level of PtdIns(4,5)P2, such as the D rized into previously established groups. Ciona inward rectifier K (Kir) channel (Fig. 5). Alterna- intestinalis (Ci)-VSP is encoded by one such novel tively, enzymatic activities can be quantified using gene.43) Ci-VSP shows homology to both the VSD of fluorescent protein sensors for PIs. Through quanti- VGICs and the tumor suppressor PI phosphatase tative measurements of enzyme substrates using PTEN. Unlike VGICs, VSP lacks a PGD. Within these sensors, it was shown that the enzyme activity VSP, a single VSD is connected to a cytoplasmic PI gradually increases over a wide range of membrane phosphatase region with similarity to PTEN.44) The potentials.55) This nature contrasts with shaper PTEN-like region of VSP contains a phosphatase voltage-dependent gating of VGICs, which requires domain (PD) with a catalytic center and a C2 activation of multiple VSDs within the channel domain, which associates with the membrane complex until pore opening (Fig. 6a). (Fig. 1). Unlike PTEN, VSP lacks a region corre- As has been well established with the VSDs of sponding to the C-terminal disordered region, which VGICs, activation of the VSP VSD occurs in several is critical for kinase-dependent regulation of enzyme steps that depend on the pairing patterns of the salt activity. The linker between the VSD and the PD bridges between the positive charges on S4 and of VSP, which we call the VSD–PD linker, consists negative charges on other helices. In fact, gating of two parts, a proximal part unique to VSP (i.e., not currents (also called “sensing currents” for charge conserved in PTEN) and a distal part that is movements of the VSD of VSP) show some delay conserved between VSP and PTEN. As in PTEN, in the peak from the initial rise.43) Consistent with the distal part of the VSD–PD linker contains this finding, voltage-evoked movements of the VSD, numerous positively charged residues able to bind measured from the fluorescence changes of an PIs. The mammalian orthologs of Ci-VSP are the PI environment-sensitive fluorophore conjugated to a phosphatases TPTE, TPIP, and PTEN2, which are specific amino acid in the VSD, show more than one highly expressed in the testis.45) component.47) These results indicate the VSP VSD The VSP VSD has an architecture similar to activates in multiple steps. other VSDs and consists of four helices, including the Given that VSP consists of a single VSD and a signature S4 containing multiple positively charged downstream enzyme region, a natural question is, 118 Y. OKAMURA and Y. OKOCHI [Vol. 95,

K+ VSP GIRK channel + Gβ/γ ΔV

PI(4,5)P PI(4,5)P2 PI(4)P 2

Membrane currents μA Reflecting PI(4,5)P2 decrease

+100 mV (VSP on) 100 Membrane 50 sec potential 50 mV 100 -60 mV -60 mV (VSP off) (VSP off) 150 -120 mV -120 mV

Fig. 5. Rapid changes in the Kir current induced by VSP-catalyzed changes in PtdIns(4,5)P2. Shown are representative current traces with voltage steps recorded under two-electrode voltage clamp in a Xenopus oocyte co-expressing an isoform of the Kir3.2 channel 50) (GIRK2d) with the G-protein O and . subunits and Ci-VSP. The G-protein subunits are necessary for activation of the Kir3.2 channel. A hyperpolarizing step from a holding potential of !60 mV to !120 mV activated inward Kir currents. After an episode of depolarization to 100 mV for 1 s, which robustly activated Ci-VSP, PtdIns(4,5)P2 levels are decreased because VSP activity cleaves the 5-phosphate. This leads to a decrease in the Kir current in the second hyperpolarizing step. Endogenous outward currents are elicited during a depolarizing step to 100 mV.

does the VSD in a particular activation state induce a processes, that are dependent on an increase in 57) specific related level or mode of enzymatic activity? PtdIns(3,4)P2. To address this issue, a VSD mutant that assumes Heterologous expression of Ci-VSP with a a stable intermediate state during VSD activation fluorescent PtdIns(3,4)P2 sensor protein (tandem was studied with zebrafish VSP.56) In this mutant, PH domain containing protein 1 [TAPP1]-derived VSD motion reaches a plateau within a certain pleckstrin homology domain (PHD) fused with green voltage range but then increases further at a higher fluorescent protein (GFP)) showed that voltage- voltage. When enzymatic activity was quantified dependent changes in the PtdIns(3,4)P2 concentra- through measurement of PtdIns(4,5)P2-sensitive Kir tion are biphasic: it increases with smaller depolari- channel activity over a wide range of membrane zations but decreases with larger depolarizations. potentials in cell attached patches, the enzymatic This result raises the intriguing possibility that the activity showed a bi-phasic pattern consistent with subreaction from PtdIns(3,4,5)P3 to PtdIns(3,4)P2 the pattern of VSD motion.56) This raises the is more prominent than other subreactions with possibility that distinct states of the VSD are coupled smaller depolarizations, whereas the subreaction with different modes of enzymatic activity. from PtdIns(3,4)P2 to PtdIns4P becomes more In addition to its voltage-dependent regulation, prominent with larger depolarizations. This idea the broader substrate preference (PtdIns(3,4,5)P3, was tested in more detail by Isacoff’s group using PtdIns(3,4)P2, PtdIns(4,5)P2) is another noteworthy the versions of Ci-VSP with the VSD trapped in feature of VSP among PI phosphatases. Notably, certain states of activation.58) Their results are regulation of PtdIns(3,4)P2 by VSP may be of consistent with a model in which partially activated biological significance, because DF1 fibroblasts states of the VSD lead to an enzyme state with a transfected with VSP show marked morphological preference for PtdIns(3,4,5)P3, whereas the fully changes, including the development of neurite-like activated state of the VSD leads to an enzyme state No. 3] Principles and diversity of voltage sensor domain proteins 119

a Voltage-gated ion channels (Kv1.2/2.1-chimera)

VSDs 4 downΔ 2 down/ 2up V ΔV 0 down/ 4up

PGD Closed Closed Opened

b Voltage-sensing phosphatase (Ci-VSP)

VSD intermediate fully up down ΔV ΔV

Enzyme inactive partially active fully active Fig. 6. Operation of voltage-sensor containing proteins. In most of voltage-gated ion channels, multiple voltage sensor domains (green) need to take up-state (activate state) until the pore is opened (upper pictures (a)). This is one of the reasons why the voltage dependence of conductance increase in voltage-gated ion channels is very sharp, as represented by voltage-dependent conductance of nerve membranes as first demonstrated by Hodgkin and Huxley. However, it should be noted that some VGICs require only one or two VSDs in the activated state to have the PGD in an open state. Lower pictures (b) show a current model of operation mechanisms of VSP. In contrast with four homologous repeats or subunits in VGICs, single VSD (green) is connected with the cytoplasmic region, which consists of the phosphatase domain (PD)(blue) and C2 domain (light green) in VSP. Upon partial activation of VSD, some phosphatase activity emerges, whereas the enzyme is fully activated upon full activation of VSD. PD has a flexible structure and its association with the membrane is critical for full activation of the enzyme when the VSD is fully activated. This second step requires a special motif, called a “hydrophobic spine” (not drawn in the diagram).60) In both cases, illustration of the motion of S4 follows the classical idea of translation motion for the simplicity of the figure, rather than ideas of transporter model and helical screw model.158)

with preference for PtdIns(3,4)P2 and PtdIns(4,5)P2. that local structural changes in PD are responsible On the other hand, through mathematical modeling, for regulating enzymatic activity. However, direct taking into account endogenous enzyme activities, evidence for such dynamic structural rearrangement and careful rapid measurements of four PIs upon of the cytoplasmic enzyme region of VSP in live cells changes in membrane potential, a simple scheme of has been difficult to be obtained due to the lack of common substrate preference among distinct states suitable methods. Most tag molecules, such as GFP, of the VSD could be fitted to the kinetics of four PI cannot be utilized for this purpose because of their species upon activation of VSP in HEK293T cells.53) bulky structure. 3-2. Mechanisms of coupling between VSD A recent study involving genetic incorporation motion and enzyme activity. Analysis of the of an environment-sensitive, fluorescent, unnatural crystal structure of an isolated cytoplasmic region of amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-ami- Ci-VSP showed distinct protein structures within the nopropanoic acid (Anap) using an orthogonal pair substrate binding pocket.52) In particular, a loop consisting of an amber suppressor tRNA and an structure, called a “gating loop,” was at a key position engineered amino-acyl-tRNA synthase enabled de- within the structure of the substrate binding pocket tection of local structural changes in the enzyme in the PD, raising the possibility that this gating loop region associated with VSD motion.59) The size of regulates the size or profile of the binding pocket and, Anap is similar to that of tryptophan. Through thus, the on and off of the enzyme. This indicates genetic incorporation of Anap, several sites within 120 Y. OKAMURA and Y. OKOCHI [Vol. 95, the gating loop (Asp-400, Phe-401, Phe-407, Gln- Mutation of the basic residues within this motif 408) were individually labeled in separate experi- markedly weakens coupling between the VSD and ments.59) Large changes in Anap fluorescence were enzyme,59),62),63) and depletion of PIs also influences detected upon membrane depolarization in all coupling, raising the possibility that by binding to mutants, indicating that the gating loop moves the VSD–PD linker, PIs may facilitate coupling dynamically during coupling with VSD motion. This between the VSD and the enzyme. Together with the study also detected changes in the fluorescence of finding that the VSD–PD linker interacts with the Anap incorporated at sites within the C2 domain, PD via a salt bridge, these results indicate that the which were believed to be static in PTEN. The speeds VSD–PD linker is a key node for interaction with of the changes in Anap fluorescence in the gating both the membrane and the PD. Additionally, loop and C2 domain were comparable, indicating potential regulation by PI of coupling between the that conformational changes for VSD-driven enzy- VSD and the downstream effector is reminiscent of matic activity are not restricted to the PD; they are the KCNQ1 and TPC1 channels, which require PI distributed over the entire enzyme region. binding for efficient coupling between the VSD and Notably, the intensity of fluorescence from PGD.30),31),33) PIs are also known to dock within a Anap introduced into the C2 domain (e.g., at Lys- space between the VSLD and PGD in TRP 555 of Ci-VSP) changed in opposite directions (early channels.64),65) However, direct evidence for physical decrease and late increase) over the course of a binding of PI to the VSD–PD linker in VSP is membrane depolarization.59),60) This indicated the lacking. enzyme has multiple activated states that are Role of the membrane interface in coupling. Given dependent on membrane potential. Recently, a that substrates for VSPs are PIs within membranes, detailed study of a K555Anap mutant revealed that it would be expected that the protein–membrane the early and late changes could be isolated through interface is crucial for the regulation of enzyme mutagenesis. Introduction of a single amino acid activity. Consistent with this idea, the C2 domain, mutation into a novel membrane interface, which which associates with the membrane, is critical for we call the hydrophobic spine, enhanced or reduced VSP enzyme activity: deletion of the domain or the late increase in Anap fluorescence. This suggested amino acid replacement within it alters enzyme the second transition (represented by the late activity.66),67) A recent molecular dynamics simula- increase in Anap fluorescence) depends on the tion and functional analysis of a heterologous hydrophobic spine, whereas the first transition expression system led to the identification of a novel (represented by the early decrease in Anap fluores- interface between the membrane and the PD, which cence) does not.60) is critical for the regulation of enzyme activity.60) The Role of the VSD–PD linker in coupling. The 20- molecular dynamics simulation of the PD highlighted amino acid VSD–PD linker is critical for coupling; two successive conserved hydrophobic residues (L284 deletion or mutation of the linker eliminates or re- and F285 in Ci-VSP) in the PD, which face the duces the voltage-regulated enzyme activity.43),61)–63) membrane. These two hydrophobic residues are The crystal structures of the VSP enzyme revealed followed by a hydrophilic residue, which destabilizes a salt-bridge interaction between a basic amino acid the membrane insertion. This structure appears to in the VSD–PD linker (R253, R254) and an acidic provide flexibility to the PD. Mutation of either of residue (D400) on the gating loop,52) and mutation of the hydrophobic residues markedly changes voltage- Asp400 markedly weakened the VSD-coupled en- dependent enzyme activity. Moreover, the magnitude zyme activity, suggesting interactions between the of the change is graded depending on the hydro- VSD–PD linker and the gating loop are critical for phobicity of the side chain of the amino acids. This the coupling between voltage-induced motion of the suggests the hydrophobicity of the hydrophobic VSD and the enzymatic activity of the PD. spine is critical for enzyme activity. This membrane Contributing to the complexity of the coupling interface is conserved in PTEN, raising the possibil- is that substrate PI can exert an allosteric effect on ity that the hydrophobic spine is crucial for innate voltage-regulated enzyme activity. Like the corre- enzyme activity but not for coupling between the sponding segment in PTEN, the distal segment of VSD and enzyme. However, substitution of an amino the VSD–PD linker contains multiple basic residues acid residue with an aromatic ring in the side chain (K252, R253, R254) in a region often called the (e.g., tryptophan) increases voltage-dependent en- PI-binding motif (PBM), which likely bind PIs.63) zyme activity but does not enhance the phosphatase No. 3] Principles and diversity of voltage sensor domain proteins 121 activity of the isolated phosphatase domain, as a VGIC was utilized for developing GEVIs.26),80) examined in vitro in a malachite green assay. This However, both the expression efficiency and volt- indicates that the hydrophobic spine is involved in age-sensitive signal are low in GEVIs designed based both the intrinsic enzyme activity and the coupling on VSDs from VGICs; consequently, these probes between the VSD and enzyme. Analysis of fluores- are not practical for monitoring the electrical cence from K555Anap with a mutation in the activities of cells. The better portability of the VSD hydrophobic spine showed that the voltage-depend- of VSP than VSDs from VGICs probably reflects ent enzyme activity correlates with the second the fact that the effector driven by the VSD is a (increasing) component of the fluorescence but not cytoplasmic structure in VSP, not a transmembrane the first (decreasing) component.60) These results structure like a VGIC. lead to three conclusions: (1) the membrane interface is critical for robust enzyme activity; (2) two stepwise 4. Ions permeate through the VSD: gating pore B transitions underlie enzyme activation upon VSD ( ) current, Hv1 and other cases motion (Fig. 6b); and (3) the second step in enzyme Within VGICs, positively charged residues on S4 activation, which is mediated by interaction of the translocate upon changes in membrane potential and hydrophobic spine with the membrane, is required for change their salt bridge partners (negatively charged robust enzyme activity. residues) on the other helices comprising the VSD, 3-3. LEGO-blocked VSD of VSP: sophisti- which passes through a constricted hydrophobic cated piece for engineering. The VSP VSD can be barrier in the middle of the electric field. This barrier swapped with another protein domain like a LEGO region faces two water crevices, one from the block to confer voltage sensitivity. Examples of extracellular side and the other from the intracellular this include VSPTEN, where the VSD of Ci-VSP side, and provides a transfer center for positively was fused to the N-terminus of human PTEN.68) charged residues in S4. The translocation of the VSPTEN exhibits voltage-dependent phosphatase positively charged residues in S4 through this path- activity toward PtdIns(3,4,5)P3. In another example, way is usually not large enough to be accompanied the VSD of Ci-VSP was transferred to a virus-derived by ion flow. However, under some conditions it is KD channel (Kcv).69) With this transfer, voltage- accompanied by ionic conductance. For instance, D 12),81) 82) insensitive Kcv became a voltage-dependent K Kv and Nav channels containing single amino channel. In a third example, voltage sensitivity was acid mutations within the helices of their VSDs conferred by transferring the VSP VSD to one or show state-dependent cation conductance (Fig. 7). two fluorescent proteins.70)–72) Using this strategy, a When a non-mutated Shaker channel VSD lacking variety of gene-encoded voltage indicators (GEVIs) the PGD is expressed heterologously in Xenopus that use the VSP VSD have been designed to oocytes, proton or monovalent cation conductance visualize electrical activities73),74) and have enabled emerges.83) These currents are called B currents or fluorescence-based detection of single action poten- gating pore currents (Fig. 7a) and differ from regular tials in CNS neurons.72),75),76) In addition, voltage ionic currents through the central pore (which are changes within fine structures, such as dendritic “, currents”). In Nav channels with mutated VSDs spines, which cannot be accessed using conventional seen in some groups of patients with hypokalemic electrophysiological methods, have been studied periodic paralysis, disruption of pH homeostasis of using ArcLight, a versatile VSP VSD-based the cytoplasm due to proton-leakage through mu- GEVI.77),78) The bioluminescent voltage probe tated VSDs likely underlies the pathophysiological LOTUS-V was also designed based on the VSP changes in skeletal muscle seen in these patients.84) VSD. Instead of a donor fluorescent protein, LOTUS- 4-1. Basic properties of Hv1. Hv1 (a.k.a. V contains a bioluminescent protein, NanoLuc, voltage sensor domain only protein, VSOP), a which is 150 times brighter than firefly luciferase.79) voltage-gated proton channel, has a VSD but lacks A common problem shared by all fluorescent GEVIs a PGD.85)–87) The VSD plays dual voltage sensing is the phenomenon of photobleaching, which makes and proton permeation roles in Hv1. The salt bridges long-term monitoring of electrical activities difficult. formed between the three positively charged residues LOTUS-V does not require illumination and so in S4 and negatively charged residues in S1 are enables long-term monitoring of cellular electrical critical for proton-selective conduction. The proper- activities with a low background signal and little ties of Hv1 currents differ from those of B currents 73) cell damage. There are cases in which the VSD of through VGIC VSDs. Hv1 shows depolarization- 122 Y. OKAMURA and Y. OKOCHI [Vol. 95,

Fig. 7. Examples of ion conductances through VSDs. a. B-current (gating pore current) through the VSD of a Shaker Kv channel R362C mutant (denoted R1C), in which the first arginine of S4 is replaced with cysteine.81) The PGD is contained in this construct, which was expressed in a Xenopus oocyte. The currents were recorded using patch clamp with 100 mM KCl, 10 mM HEPES, and 1 mM EDTA, pH 7.1 in both the bath and patch pipette solutions. Currents were measured while stepping from a holding potential of !110 mV to test potentials ranging from !200 to D60 mV in 20-mV steps, followed by repolarization to !100 mV. Red traces indicate B currents evoked by hyperpolarization; black traces are currents through the canonical pore. Some inward currents (black inward traces) appear with small depolarizations because of a negative shift in the I-V curve of the R1C mutant. Transient large outward currents are through the canonical pore. The transient profile of the outward current is due to the fast inactivation of Shaker channels retaining the N-terminal inactivation ball. B currents (red) do not exhibit such inactivation, which is consistent with the view that ions flow is through a permeation pathway (gating pore of VSD) separate from the canonical pore. b. Voltage-gated proton current 159) through mouse Hv1 heterologously expressed in a HEK293T cell. Shown are a family of traces evoked by test pulses stepped from a holding potential of !60 mV to a level ranging from 10 mV to 130 mV in 20-mV increments for 3 s. The bath solution contained (in mM) 180 HEPES, 75 N-Methyl-D-glucamine (NMDG), 1 MgCl2, 1 CaCl2 (pH 6.9). The internal solution contained 183 HEPES, 65 2D NMDG, 3 MgCl2, 1 EGTA (pH 7.0). pH was adjusted using methanesulfonate. c. Hyperpolarization-activated Ca currents in a HEK293T cell heterologously expressing the VSD from an ascidian CatSper channel subunit, Ci-CatSper3.134) The structure downstream of the VSD including PGD is truncated in this construct. The external solution contained (in mM) 150 NaCl, 2 CaCl2,10 HEPES (pH 7.4). Internal solution contained 130 CsCl, 1 EGTA, 50 HEPES, pH 7.4. Step pulses were applied from a holding potential of !10 mV to a level ranging from D50 mV to !150 mV in 20-mV increments. The trace at !150 mV is shown in red. induced activation of conductance, whereas most of that the mechanism of proton conduction differs from B currents are activated by hyperpolarization. In that underlying B currents through VGIC VSDs. addition, Hv1 tail currents show clear voltage- The most critical characteristic of Hv1 is its high 6 dependent kinetics (Fig. 7b), whereas the offset of B proton selectivity; Hv1 is at least 10 times more currents is not associated with a clear tail current. conductive for HD than NaD. Selectivity lower than Amino acid mutation of Hv1 makes the channel that does not make sense for a “proton channel,” conductive upon membrane hyperpolarization, mim- given that proton concentrations at the pH of normal icking the B current without affecting the depolari- serum (pH 7.4) corresponds to less than 100 nM, zation-activated outward current.88) This indicated which is about 10!6 times lower than the concen- No. 3] Principles and diversity of voltage sensor domain proteins 123

S6 S4 S5 helical linker abhelical linker S4 S6 S5 + & + + PI + + VSD PGD + VSD + PGD + + S1 S1 +

S4 S6 S5 c + + S1-S1 PGD d VSD + S4-S4 S1 + proton permeation + + through VSD VSD + + VSD Cytoplasmic domains + + S1 S1 e + S4 + VSD + + S1 PI S4-coiled coil

helical linker? enzyme

Fig. 8. Various types of coupling with the VSD among VGICs and voltage sensor domain proteins. a. In domain-swapped VGICs, a complex of the helical linker between S4 and S5 with a part of S6 close to the cytoplasm is critical for transmitting the information of S4 motion to the PGD, leading to pore gating. S4 has a signature alignment of amino acids: several positively charged residues are situated periodically with intervening hydrophobic residues along the helix. b. In domain-swapped, PIP2-sensitive VGICs (e.g., KCNQ1), binding of PI to the S4–S5 linker is critical for coupling. c. In domain-nonswapped VGICs, the VSD couples to the PGD via a linker that is shorter than the linker in domain-swapped VGICs. Both the VSD and PGD interact with cytoplasmic domains that recognize small ligand molecules. Splitting the whole polypeptide within the linker does not eliminate coupling, suggesting helix–helix interaction within the membrane is more important for coupling. d. In Hv1, which lacks a PGD, two VSDs interact through three interfaces: S1–S1, S4–S4, and S4-coiled coil. Dotted arrows indicate the dimer interaction interfaces. Arrows indicate the interaction between S4 and the coiled coil. Within the 3D protein structure, these sites are next to each other when the dimers are in certain conformations. e. VSP lacks a PGD but contains a PTEN-like PI phosphatase. Dephosphorylation of several species of PIs is induced by depolarization-activated motion of the VSD. The upward motion of S4, which harbors several positively charged residues, induces a conformational change in the enzyme. The VSD-phosphatase linker is critical for this coupling, though the structure of the linker remains unknown (dotted box). tration of NaD ions in normal serum. The molecular model). In another model, protonation of amino acid mechanism of proton permeation through Hv1 has side chains underlies shuttling of protons through 89),90) been the target of hot debate. Proposed ideas for the channel. D112 in human (h)Hv1 (corresponding proton-selective permeation can be broadly catego- to D108 of mouse (m)Hv1) is known to be critical rized into two models.91) In one model, a water wire is for proton-selective permeation and to form a salt present in the form of a wedge from both sides of the bridge with an asparagine residue in S4. This membrane, and water molecules stably sitting in the asparagine residue is thought to be a potentially middle of the constricted area complete the water titratable residue for proton conduction.92) Other wire for proton conduction (so called “frozen water” signature characteristics of Hv1 are voltage-depend- 124 Y. OKAMURA and Y. OKOCHI [Vol. 95,

ent gating regulated by the pH difference across These studies in Hv1-KO mice supported the model the membrane, gating inhibition by external zinc ions that Hv1 maintains NADPH oxidase activity by (as described in a later section),90) and high temper- regulating intracellular pH and membrane potential ature sensitivity.89),93) Basic properties of voltage- for ROS production. The regulation of ROS produc- gated proton currents have been explained in earlier tion by Hv1 has also been observed in other immune reviews.87),89),90),93),94) cells, including T lymphocytes,105) B lymphocytes106) 107) Unique among VGICs, Hv1 shows dimeric and macrophages. However, the detailed mecha- stoichiometry, with two VSDs within the protein nisms underlying the regulation of ROS production complex. Hv1 has a C-terminal cytoplasmic region in these cells remain unclear. 95) with a coiled coil structure that has the ability to The function of Hv1 in neutrophils appears to be form a dimer. Coimmunoprecipitation96) and fluores- multimodal. Hypochlorous acid (HOCl), a ROS, is 96),97) cence labeling studies showed that Hv1 functions generated from hydrogen peroxide (H2O2) by myelo- as a dimer within the membrane. Nonetheless, peroxidase (MPO). MPO is released by cells into the monomeric Hv1 protein lacking the C-terminus still extracellular space through exocytosis (degranula- 96),97) functions as voltage-gated proton channel, tion). Hv1-KO neutrophils release more MPO than indicating that most of the hallmark features of the wild-type neutrophils, resulting in excess HOCl channel do not require dimerization. production.108) The enhanced degranulation of The crystal structure of mouse Hv1 in a closed MPO-containing granules is suppressed by the state was solved as a chimeric protein (mHv1cc) with NADPH oxidase inhibitor diphenyleneiodonium the S3–S4 region from Ci-VSP and the coiled coil chloride or the KD ionophore valinomycin,108) which region from a yeast transcription factor, GCN4.98) suppresses membrane depolarization. This suggests Comprising the entire structure are four helices that that Hv1 inhibits degranulation of MPO-containing are slightly oblique and wider at the lower side, like a granules in neutrophils through the regulation of closed umbrella. S4 runs straight across the mem- membrane potential changes, which are induced by brane, extending its helical structure to the cytoplas- NADPH oxidase activation. mic coiled coil. Microglia, resident immune cells in the brain, 4-2. Summary of the biological functions of not only protect the brain from invading pathogens, Hv1. Hv1 is expressed in the immune cells of many but also clear dead cells and regulate the micro- 109) species from teleosts to humans, the epithelium of environment. The impact of Hv1 on ROS produc- the respiratory system in mammals, and sperm and tion in microglia differs from that in other immune cancer cells in humans. The Hv1-encoding gene, cells in that microglia isolated from Hv1-KO mice HVCN1, has also been found in the genomes of exhibit increased ROS production.110) The distribu- marine plankton99) and insects,100) but absent in tion of p67, a NADPH oxidase subunit, and actin 87),90) Caenorhabditis elegans and Drosophila. Before dynamics are altered in Hv1-KO microglia, which 110) identification of the Hv1 molecule, voltage-dependent may underlie the increase in ROS production. proton conductance was studied extensively with This idea is consistent with the observation that reference to its role in regulating reactive oxygen facilitating actin filament formation using jasplaki- species (ROS) production in phagocytes.89),101) nolide increases ROS production in wild-type micro- NADPH oxidase is electrogenic, and the oxidase glia,110) as well as with studies showing that local- transfers electrons from cytoplasmic NADPH to ization of cytosolic NADPH oxidase subunits and oxygen outside membrane to generate ROS. In this ROS production are affected by actin dynam- 111),112) reaction, protons are released into the cytoplasm. ics. Hv1 probably inhibits ROS production The electron transfer and proton release cause through effects of actin dynamics, although the membrane depolarization and acidification of the detailed mechanism remains unknown. These emerg- cytoplasm. Hv1 channel activity is believed to cancel ing findings indicate that the function of Hv1 in ROS these effects to maintain NADPH oxidase activity.89) production is more complex than previously thought. Studies using knockout mice lacking HVCN1 (Hv1- Among various cell types, it is likely that Hv1 KO) revealed that Hv1 is necessary to maintain ROS contributes to cellular function through diverse production at a high level in neutrophils.102),103) It mechanisms that depend on the physiological or was also revealed that Hv1 in neutrophils act to pathophysiological context. inhibit acidification of the cytoplasm and excess Although the physiological functions of Hv1 have 104) depolarization upon NADPH oxidase activation. been investigated extensively in Hv1-KO mice, the No. 3] Principles and diversity of voltage sensor domain proteins 125

118) actions of Hv1 in humans remain unclear. Recently, within the same dimer. a potent and highly selective human Hv1 inhibitor, How is such coupling between two protomers Corza6 (C6), was developed based on the structures achieved? For cooperative gating of Hv1, it has been of peptide toxins acting on different VGICs.113) shown that three sites of intersubunit contact (S1, Inhibition of Hv1 activity in human neutrophils and S4, and the coiled coil) between the two protomers sperm using this reagent shed light on the physio- are critical (Fig. 8d).119) logical roles of Hv1 in sperm maturation critical for S1–S1. The results of cysteine mutagenesis fertilization, so-called capacitation, as well as its experiments suggested that the external ends of the enhancement of ROS production in neutrophils.113) S1 segments are positioned next to each other within 120) 4-3. Twin VSDs work together to regulate Hv1 dimers through highly efficient crosslinking. proton conduction in Hv1. A remarkable feature of VCF measurements of VSD motion within Hv1 Hv1 channel gating is cooperativity within the dimer; showed that cooperativity-dependent fluorescence status of one monomer influences the conformational changes were no longer observed after mutation of change of the other monomer in the dimer. Based on acidic residues at the external end of S1 in sea squirt 117) this cooperativity, dimeric Hv1 shows sharper voltage Hv1 (CiHv1). Open-state coupling between the dependence than the monomer, although it is two protomers, as determined from their sensitivity shallower than that of most of VGICs such as Kv to an open channel blocker, was also diminished by 118) and Nav, which play principal roles in excitable cells. similar mutations in hHv1. Cooperative gating of Hv1 was demonstrated by Coiled coil. Biochemical analyses and analysis of findings obtained in four types of studies employing the crystal structure of the isolated coiled coil motif heterologous expression systems. First, electrophy- showed that this motif in the C-terminal cytoplasmic siological analysis of channel activation and deacti- region of Hv1 has innate activities related to dimer vation showed a delay in the activation phase of the assembly,95),121) and that amino acid mutations dimeric channel, whereas a mono-exponential rise within the coiled coil altered the strength of dimer without a delay was seen in the monomeric chan- assembly and affect channel gating.121) In addition, nel.114),115) In addition, measurements of the limiting electrophysiological characterization of cooperative slope of activation with small increases in depolariza- gating and western blotting analyses of cross-linking tion enabled estimation of the magnitude of the showed that S4 forms a continuous helix with the translocated charges per channel, which, in dimeric cytoplasmic coiled coil.122) Mutation of the linker channels, were double those of monomeric chan- region between S4 and the coiled coil modified the nels.116) Second, analysis of gating kinetics by label- extent of cooperative gating, supporting the idea ing specific amino acids with an environment- that the S4–coiled coil helical structure provides a sensitive fluorophore (voltage clamp fluorometry core structure for dimer interaction. The continuous [VCF]) enabled detailed analysis of the activation helical nature of the S4–coiled coil region was and deactivation process in VSDs.117) In contrast to confirmed by two studies of protein structure: an measurement of ionic currents, which are the final analysis of the crystal structure of the mHv1 chimeric 98) outcome of a conformational change in an ion protein mHv1cc and an examination of the channel, VCF permits analysis of conformational structure based on electron paramagnetic resonance changes associated with transitions to partially (EPR) and in silico analyses.123) activated, but nonconductive, states. Experiments S4–S4. Guided by the model of the dimeric 98) using VCF showed that activation occurs in multiple structure of mHv1cc, the interaction between the steps and only the last transition of the voltage two S4s running in parallel through the membrane sensor is coupled to pore opening. Careful comparison was closely examined.124) A tryptophan residue in of the kinetics of fluorescence changes with the S4 is highly conserved among Hv1 orthologs, and a activation kinetics of proton currents led to the idea molecular dynamics simulation predicted that it is that there is intimate cooperativity between dimers positioned at the dimer interface and that the during the final transition for pore opening.117) Third, tryptophans in the two protomers are close to one a seminal study using the open pore blocker 2-GBI another. Mutation of the tryptophan accelerates showed that the kinetics of the blockade of two deactivation, and it was suggested that S4–S4 individual pores differ between monomeric and interaction occurs during deactivation but not dimeric channels, indicating that the state of one during activation.124) This was consistent with the subunit influences the state of the other subunit idea that state transition of the VSD occurs through 126 Y. OKAMURA and Y. OKOCHI [Vol. 95, multiple conformations during activation and deac- The Zn2D binding sites are conserved between 125) tivation. mouse and human Hv1 orthologs. However, in The crystal structure of mHv1cc in the closed zebrafish and the African clawed frog, the binding state showed that the S4 segments are close to each sites are not fully conserved.127) Electrophysiological other, but the S1 segments are not. Perozo’s group analyses showed that zebrafish (Dr)Hv1 is resistant 2D 127) used EPR analysis to study hHv1 and modeled its to 10 µM Zn , which strongly inhibits mHv1 123) 85) dimeric structure. Notably, the structure re- currents. Of note, the low sensitivity of Dr-Hv1to mained dimeric even after deletion of a large section Zn2D appears to be related to the serum Zn2D of the dimeric coiled coil, which suggests the VSD concentration, which is around 15 µM in mice and itself has some capacity for self-assembly as a dimer. humans,128) but is 10 times higher in zebrafish and Importantly, this structure showed the S1 segments clawed frog.127) This suggests the low sensitivity of 2D to be close to one another, unlike in the model based Dr-Hv1toZn is necessary to ensure adequate ROS 98) on the crystal structure of mHv1cc, which was production by zebrafish neutrophils. consistent with results from VCF117) and electro- It has been suggested that the Zn2D sensitivity of 118) physiological studies. It is possible that these two Hv1 is physiologically significant in human sperm, 129) structures represent slightly different states during which abundantly expresses Hv1. Human seminal activation and that a subtle change in the angle plasma contains a high concentration of Zn2D across individual helices may cause the neighboring (92mM),130) which diffuses away within the female 131) S1 segments to change the state of their interaction. reproductive tract. This supports idea that Hv1is 2D To summarize, the two Hv1 protomers interact inhibited by Zn within the testis, but the dilution with each other at distinct contact sites on S1, S4, of Zn2D within the female reproductive tract leads to and the coiled-coil domain. The function of each site Hv1 activation promoting hyperactivation of sperm may vary during cooperative gating, possibly reflect- (capacitation). It has also been suggested that the 2D ing the particular state during activation/deactiva- Zn sensitivity of Hv1 has physiological relevance tion of the VSD. to host defense by granulocyte-macrophage colony- 132) 4-4. Zinc sensitivity of Hv1: structural basis stimulating factor-treated macrophages. and biological significance. Another hallmark 4-5. More examples of ion conductance 2D property of Hv1 is its high Zn sensitivity. External through the non-mutated VSDs. In addition to zinc at submicromolar levels inhibits Hv1 gating. As Hv1, examples of ion conduction through non- the Zn2D concentration increases, activation becomes mutated VSDs include CatSper (cation channel of slower, and the activation voltage is shifted to more sperm), a Ca2D-permeable channel complex consist- positive levels, which indicates Zn2D is not a pore ing of multiple subunits specifically expressed in blocker but a gating modifier.89) An early muta- spermatozoa. CatSper is the major route for Ca2D genesis study showed that two histidines (H140 and influx into sperm. The , subunit of CatSper shows H193 in hHv1, corresponding to H136 and H189 in remarkable similarity to VGICs, and a defect in the 133) mHv1) facing the external side of the channel play , subunit leads to infertility. Our recent study important roles in determining Zn2D sensitivity.86) showed that a VSLD from the , subunit of sea squirt 2D The crystal structure of mHv1cc showed that two CatSper3 mediates a slowly activating Ca current other amino acids also contribute to the Zn2D upon membrane hyperpolarization in two heterolo- 98) sensitivity of mHv1: E115 and D119. Although gous expression systems: Xenopus oocytes and individual mutations of E115 or D119 had no effect HEK293T cells (Fig. 7c).134) Unlike the B current on Zn2D sensitivity, double mutation abolished the (or gating-pore current) derived from the Shaker sensitivity to Zn2D. A recent attenuated total channel VSD,83) this Ca2D current persists with the reflectance-Fourier transform infrared spectroscopy expression of the full-length, PGD-containing pro- analysis in combination with quantum chemical tein. Moreover, Ca2D influx was detected upon calculation provided a detailed profile of the Zn2D hyperpolarization to !60 mV, raising the possibility coordinating mechanism. Zn2D is coordinated by N1 that Ca2D influx through the VSD could occur within (Ni) of the neutral imidazole of histidine, the anionic a physiological range of membrane potential. The carboxylate of an acidic residue, and one or two VSD from mammalian CatSper3 exhibits similar water molecules. The anionic carboxylate of the characteristics.135) However, heteromeric assembly of acidic residue binds Zn2D in the monodendate mode, multiple CatSper subunits is known to be essential and either one or two histidines binds Zn2D.126) for normal sperm motility,136) indicating that possible No. 3] Principles and diversity of voltage sensor domain proteins 127

Ca2D influx through the VSD is not the dominant the sea urchin, Strongylocentrotus purpuratus, the pathway through the CatSper channel. Whether VSLD exhibited gating currents (corresponding to such conductance through the CatSper VSD actually the voltage-dependent charge motion associated with occurs in spermatocytes remains unknown. the VSD motion) in a heterologous expression system Vriens et al. suggested that TRPM3 has two and conferred voltage-sensitivity to its sodium- distinct ion permeation pathways, which are trig- proton exchanger activity.141) It will be intriguing gered by specific stimuli.137) Ligands such as the to know how the effector domain (NHE domain) in neurosteroid pregnenolone sulfate (PS), heat, or SpSLC9C1 is driven by the C-terminal VSLD. nifedipine activate canonical outwardly rectifying TRP channels are a large superfamily of cation currents, whereas hyperpolarization-activated in- channels that show significant homology to VGICs. wardly rectifying currents are induced in the They include TRPV, TRPM, TRPC, TRPA, TRPN, combined presence of PS and clotrimazole. Charac- TRPML, and TRPP channels and are activated by teristics of the second current that differ from the diverse chemical and physical stimuli. The protein canonical current include (1) strong inward rectifi- structure of the transmembrane region of TRPV1, cation, (2) resistance to Ca2D-dependent desensitiza- the heat-sensitive TRP channel, is remarkably similar tion, (3) low permeability to Ca2D, (4) low sensitivity to two features of domain-swapped VGICs.142),143) to La3D blockade, and (5) resistance to amino acid First, TRPV1 shows domain-swapped positioning of mutation and cysteine modification at the central a voltage sensor-like domain (VSLD) and the PGD. pore. The noncanonical ionic conductance through Second, the clear ,-helical linker present between the TRPM3 may underlie drug-induced pain sensation, VSLD and PGD contains a flex point within S5.143) because clotrimazole potentiates the TRPM3-medi- On the other hand, the cytoplasmic half of the core ated responses of dorsal root ganglion neurons region contains a number of hydrophobic residues in vitro and exacerbates PS-induced pain in animal packed into a region corresponding to the water- behavior experiments.137) A recent study also showed filling crevice of the VGIC VSD, which can be that the noncanonical conductance is affected by accessed from the cytoplasm (between S3 and S4). mutation of S4 as well as S1 and S3 of the VSLD, This suggests the VSLD is a rigid structure in indicating that the noncanonical current is through TRPV1, which is consistent with the finding that, the VSLD.138) Whether other TRPM subtypes have a in yeast, nonbiased random mutagenesis in the S4– similar capacity for ion conduction through the VSD S5 linker, but not S1–S4 region, frequently produces remains to be established. a gain-of-function phenotype.144) Given that the S4–S5 linker forms a pocket to bind capsaicin, a 5. Other functions of VSD TRPV1 activator, the stable nature of the VSLD in Proteins containing VSD-like structures contin- TRPV1 ensures that it operates as a stable anchor 142) ue to emerge. A protein called HVRP1 (Hv1-related upon ligand-induced channel activation. Notably, protein 1) is abundantly expressed in cerebellar S4–S5 is flanked by another conserved helix down- granule neurons and has an amino acid sequence stream of the PGD, the TRP motif, which is 139) similar to Hv1. Its VSLD, which consists of four associated with two N-terminal structures: the transmembrane helices, is flanked on the C-terminal ankyrin-repeat and pre-S1 helix. Conservation of side by a coiled coil motif and an extended region the TRP motif among TRP channels suggests a that shows no homology to known proteins. Voltage network involving the TRP motif, S4–S5 linker and clamp fluorometry showed that the VSLD of HVRP1 PGD is central to integration of physical and chemi- senses membrane potential. What kinds of down- cal signals toward pore opening. Liao et al.143) stream cascades are activated by voltage via HVRP1 proposed that the similar but distinct structures of remain a mystery. A VSD-like domain has also been VGICs and TRP channels reflect distinct gating found in a sperm-specific isoform of the NaD-HD modes (voltage versus chemical signals). exchanger (sNHE or SLC9C1).140) This VSD-like Within the structure of TRPV2, the VSLD domain consists of four transmembrane segments consists of four helices (S1–S4) containing multiple (S1–S4-like helices) situated downstream of the NHE aromatic residues, suggesting a rigid, immobile region. In addition, there is a consensus sequence for structure. A recent study showed that the structure a putative cyclic nucleotide-binding domain in the of another TRPV channel, TRPV6, is domain C-terminus. In a recent study of the molecular nonswapped.145) It will be interesting in the future functions of SpSLC9C1, the sNHE homolog from to determine the mechanisms by which chemical and 128 Y. OKAMURA and Y. OKOCHI [Vol. 95, physical signals are sensed by TRPV channels and potential, whether this voltage dependence is derived how they differ between domain-nonswapped and from the VSD-like structure in RyRs is an important domain-swapped channels. issue. A structure with some similarity to the VSD In the TRPML channel, a voltage-sensitive has also been found in the TRIC channel, a cation Ca2D-permeable channel in endosomes/lysosomes, channel in the ER membrane.151) TRIC channels PtdIns(3,5)P2 regulates gating. The recently describ- are trimeric with a pore in each individual protomer. ed cryo-EM structure of the TRPML channel One of the transmembrane helices, M4, contains revealed a unique cytoplasmic region, termed the three conserved positively charged residues facing one mucolipin domain, which connects the VSLD to the side of the protein,151) which is reminiscent of the PGD. The mucolipin domain consists of cytoplasmic pattern in S4 of the VSD. Given that TRIC channels ,-helical stretch spanning S2, S3 of the VSLD, and show clear voltage-dependence, whether the positive S6 of the PGD. Docking of PtdIns(3,5)P2 to the S2 charges in M4 contribute to voltage sensing is an side of this domain, beneath the VSLD, imposes a intriguing question. pulling force on S6.65) This is in contrast to VGICs, where coupling between the VSD and PGD takes 6. Hierarchy of molecular organization involving place mainly within the membrane. the VSD in cellular physiology As mentioned, in many TRP channels the VSLD In its most basic form, the VSD operates as an is not mobile, but instead appears to operate as a ion channel by itself. In Hv1, two protomers interact scaffold for PGD gating and is connected to a bulky with one another to exhibit cooperative gating. In cytoplasmic region as well as to a ligand docking VSP, one VSD regulates an intrinsic enzyme. In structure involving the S4–S5 linker. In TRPM VGICs, one channel protein complex consists of four 146) channels, S4 underlies voltage-sensitive gating. VSDs and four PGDs. At a higher level, Cav1.1 Within the VSLD in TRPM subfamily members, interacts with the RyR. In that case, the stoichiom- one basic amino acid in S4 is highly conserved at a etry of the Cav1.1-RyR complex is 4:1, with a single position corresponding to one of the basic amino acid Cav1.1 binding to one subunit within the tetrameric residues in S4 within VGICs.146) TRPM4 gating is RyR.152) In other words, a complex consisting of one 2D 147) known to be regulated by cytoplasmic Ca . A RyR with four Cav1.1 channels contains 16 VSDs. recent cryo-EM study of TRPM4 with and without In peripheral neurons, Cav2.2 directly interacts with Ca2D showed that Ca2D binds within an aqueous SNARE complex, and a voltage-dependent confor- pocket in the VSLD and that its binding induces the mational change in Cav2.2 regulates SNARE complex shift in the position of the side chain of an arginine, without requiring Ca2D influx. This was identified as which changes its partner amino acid for salt the phenomenon underlying calcium independent, bridging.148) However, whether this positive charge voltage dependent secretion.153) on S4 is located in the focused electric field remains Four-repeat type VGICs, including Cav and unclear, given that the upper half over the arginine is eukaryotic Nav, have long been thought to operate as packed with hydrophobic side chains. The structure monomers. No evidence has yet been presented for of another weakly voltage-sensitive TRPM subfamily cooperativity mediated through physical contact member, TRPM8, was also solved using cryo-EM.149) among clustered, multiple channels, except perhaps Surprisingly, this structure lacked an obvious S4–S5 the pyramidal shape of clusters of Cav1.1-RyR linker but showed a pattern of domain-swapping, complexes in skeletal muscle. On the other hand, 2D D which is in sharp contrast to the Ca -activated K Cav1.3 and Nav1 were recently reported to exhibit channel Slo1. In TRPM8, S4 is long and straight and cooperativity through physical interaction among connects to S5 to form a domain-swapped conforma- individual channels on cell membranes.154),155) This tion. For both TRPM4 and TRPM8, it remains suggests VGICs may transmit voltage-dependent unclear whether interactions among helices of the information through interaction with binding part- PGD and VSLD are mediated via helix–helix ners (homologous or heterologous interactions) or by interaction or a pulling motion along the helices. forming higher-order signaling complexes. The cryo-EM structure of the ryanodine receptor (RyR) revealed that the transmembrane segments 7. Conclusions and future perspectives resemble the structure of the VSD in VGICs, despite In this review, we described VSD functions in little homology at the amino acid sequence level.150) VGICs and in two VSD proteins that lack a PGD. Because RyRs are known to be sensitive to membrane VGICs employ multiple mechanisms for coupling to No. 3] Principles and diversity of voltage sensor domain proteins 129 the VSD, including interaction among transmem- ion channel,117)–119),121),122),156) but its actual physio- brane helices and mechanical motion of the helix logical significance remains unclear. To understand linker between the two domains (Fig. 8a, 8b, 8c). how the molecular design of these VSD proteins is Unraveling the mechanisms of VSD-PGD coupling optimized for their physiological function, more in VGICs will be important for understanding the coherence between studies of the proteins themselves pathophysiology of diseases caused by mutation or (such as biophysical characterization in heterologous alteration of VGICs as well as for drug development. expression systems or structural studies of their In VSP, the VSD is coupled to an enzyme, and the molecular nature) and elucidation of their molecular linker and the membrane interfaces are critical for behavior within native cells in physiological contexts this coupling (Fig. 6). In Hv1, the VSD itself operates will be necessary. as both a channel pore and voltage sensor. Two VSDs behave as a single unit through tight interaction that Acknowledgements is dependent on multiple interfaces, including trans- We would like to thank Dr. Jianmin Cui for membrane helices and the cytoplasmic coiled coil helpful comments and careful reading of the manu- (Fig. 8d). script. We thank Ms. Yuka Jinno, Dr. Akira Comparison between VGICs and the two VSD Kawanabe, and Dr. Souhei Sakata for help with the proteins lacking PGDs provides a unique opportunity artwork of the figures, and lab members and team to gain insights into the common mechanisms of VSD members of the Atsushi Nakagawa CREST project dynamics and VSD-effector coupling. For example, (JPMJCR14M3) for helpful discussions. We would the helical linker downstream of S4 is critical for also like to thank our many collaborators for all their downstream signaling in VGICs, VSP and Hv1. The contributions over the years, which have enabled us mechanisms operating in VSD-containing proteins to write this review. This work is supported by may also be shared by other sensors, such as light- Grants-in-Aid from the Japan Society for the sensitive proteins. In addition, these findings will be Promotion of Science (JSPS) (JP21229003 to Ya. useful to strategically devise better molecular tools O. and Yo. O., JP25253016 to Ya. O., JP16H02617 for imaging membrane voltage or for manipulating to Ya. O. and Yo. O., 15K08175 to Yo. O.), cell signals. Ministry of Education, Culture, Sports, Science, Finally, we stress that there is a gap between the and Technology (MEXT) (JP24111529, biophysical mechanisms of VSD-effector coupling JP26111712, JP15H05901 to Ya. O.), and Core and the physiological functions of VSD-containing Research for Evolutional Science and Technology proteins. For example, little is known about the (CREST, JST) (JPMJCR14M3). physiological function of VSP in any animal species. 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Profile

Yasushi Okamura was born in Tokyo in 1960 and graduated from the School of Medicine, the University of Tokyo and obtained his license as a medical doctor in 1985. He went to the Graduate School of Medicine, the University of Tokyo and received his Ph.D. degree in 1989. After working as a postdoctoral fellow at the State University of New York at Stony Brook between 1989 and 1990 under the supervision by Dr. Gail Mandel (currently a researcher at the Howard Hughes Medical Institute and Professor at the Vollum Institute, Oregon Health and Science University), he was a lecturer at the Department of Neurobiology, Institute for Brain Research, Faculty of Medicine, the University of Tokyo until 1995. He was senior researcher and group leader of the Biomolecular Engineering Department, National Institute of Bioscience and Human-Technology, Agency of Industrial Sciences and Technology in Japan between 1995 and 2001. In 2001, he was appointed as a full professor at the Okazaki Institute for Integrative Bioscience in Okazaki. Since 2008, he has been a professor at the Graduate School of Medicine, Osaka University. For a long time, he has been working on ion channels, in particular their structure–function relationships and regulation of expression. He pioneered a new research field of voltage-evoked cell signals by his discovery of a novel class of membrane proteins that have voltage sensor domains but lack an authentic pore domain.

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Yoshifumi Okochi was born in Aichi in 1973 and graduated from the School of Agriculture, Hokkaido University. He went to the Graduate School of Science, Nagoya University and received his Ph.D. degree in 2005. He worked as a postdoctoral fellow at the Okazaki Institute for Integrative Bioscience in Okazaki from 2005 to 2008 under the supervision by Dr. Yasushi Okamura. Since 2008, he has been an assistant professor at Graduate School of Medicine, Osaka University. He has been working on ion channel functions at both the cellular and whole body levels.